Measurement of primary DNA damage in individual cells from humans and mice using the alkaline single cell gel electrophoresis assay

Sammanfattning: Primary DNA damage is considered to be an important step in the development of tumours and itmay also serve as a biomarker of genotoxic stress. Measurement of DNA strand breaks in peripherallymphocytes can yield useful information about the suitability of these cells for biomonitoring purposesand subsequently risk assessment. Single cell gel electrophoresis (comet) assay is a sensitive and rapidmethod for detection of DNA strand breaks in individual cells, both in vivo and in vitro. Embedding ofsingle cells in agarose gel, followed by lysis in alkali and electrophoresis causes DNA fragments toleave the nucleus and migrate in the electric field to give rise to 'comets' which can ideally be evaluatedby computerised image analysis. The aim of this thesis was to obtain data to assess the potentialusefulness of the comet assay for human biomonitoring of genotoxic exposure using peripherallymphocytes from both experimental animals and humans.Studies in mice showed that repeated exposure of mice to chlorobenzene, induced DNA damagein peripheral lymphocytes but not in bone marrow cells. Significant and dose-related DNA damage wasobserved only in the liver cells after three days of benzo(a)pyrene exposure. Styrene and styrene oxideinduced DNA damage in the all cell types investigated (lymphocytes, liver, kidney and bone marrowcells) 4 h after the exposure. A decrease in the degree. of DNA damage was observed with time in all celltypes investigated. Cyclophosphamide showed a clear dose-response effect in all types of cells examined(lymphocytes, liver, kidney and bone marrow cells).Studies in humans showed that the basal level of primary DNA damage in peripherallymphocytes from healthy volunteers was fairly constant over 5 months, although intra- andinterindividual variations were present. In a clinical study, DNA damage in peripheral lymphocytesfrom 17 women with breast cancer treated with cytostatic agents was clearly enhanced especially 16-21h after exposure. The interindividual variations in the basal level of DNA damage could not be relatedto the patients' previous experience of chemotherapy. Interindividual variations in DNA damage wereobserved also after the treatment. In a field study, the level of DNA damage in peripheral lymphocytesfrom 35 sewage workers suspected of exposure to genotoxic agents showed no increase when comparedwith 30 age-matched referents without occupational exposure to sewage.Although not all types of genotoxic exposures should be expected to result in DNA strand breaks in peripheral lymphocytes the semiquantitative assessment of primary DNA damage with the comet assay using computerised image analysis could be a valuable complement to currently used approaches to detect genotoxic exposure.